Chronic centrosome amplification PNAS PLUS without tumorigenesis Benjamin Vitrea,1,2, Andrew J. Hollanda,1,3, Anita Kulukianb,1, Ofer Shoshania, Maretoshi Hiraic, Yin Wanga, Marcus Maldonadoa, Thomas Choa, Jihane Boubakera,4, Deborah A. Swingd, Lino Tessarollod, Sylvia M. Evansc, Elaine Fuchsb, and Don W. Clevelanda,e,5 aSan Diego Branch, Ludwig Institute for Cancer Research, La Jolla, CA 92093; bHoward Hughes Medical Institute, Laboratory of Mammalian Cell Biology and Development, The Rockefeller University, New York, NY 10065; cSkaggs School of Pharmacy, University of California at San Diego, La Jolla, CA 92093; dMouse Cancer Genetics Program, National Cancer Institute, Frederick, MD 21702; and eDepartment of Cellular and Molecular Medicine, University of California at San Diego, La Jolla, CA 92093 Contributed by Don W. Cleveland, October 2, 2015 (sent for review July 6, 2015) Centrosomes are microtubule-organizing centers that facilitate bi- cation in nontransformed human telomerase-expressing (hTERT) polar mitotic spindle assembly and chromosome segregation. Rec- RPE-1 cells triggers a p53-dependent cell cycle arrest (15), ognizing that centrosome amplification is a common feature of whereas driving centrosome amplification in mice brain leads aneuploid cancer cells, we tested whether supernumerary centro- to a developmental loss of neural stem cells by p53-dependent somes are sufficient to drive tumor development. To do this, we apoptosis (16). constructed and analyzed mice in which centrosome amplification More than a century ago, Boveri suggested a link between ac- can be induced by a Cre-recombinase–mediated increase in expres- quisition of too many centrosomes and tumorigenesis (17). Nev- sion of Polo-like kinase 4 (Plk4). Elevated Plk4 in mouse fibroblasts ertheless, whether and how centrosome amplification impacts produced supernumerary centrosomes and enhanced the expected mammalian tumor development remains untested. Here we have mitotic errors, but proliferation continued only after inactivation of developed a mouse model in which centrosome amplification can the p53 tumor suppressor. Increasing Plk4 levels in mice with func- be induced by Cre-recombinase–mediated elevation in Plk4 ex- tional p53 produced centrosome amplification in liver and skin, but pression. In the presence of the p53 tumor suppressor, widespread this did not promote spontaneous tumor development in these tis- elevation of Plk4 drove the production and accumulation of too CELL BIOLOGY sues or enhance the growth of chemically induced skin tumors. In the many centrosomes in liver and skin cells, but this did not accelerate absence of p53, Plk4 overexpression generated widespread centro- tumorigenesis. Chronic elevation of Plk4 levels in mice without some amplification, but did not drive additional tumors or affect functional p53 produced widespread accumulation of cells with development of the fatal thymic lymphomas that arise in animals centrosome amplification. Even here, however, centrosome am- lacking p53. We conclude that, independent of p53 status, supernu- plification did not drive new tumors or affect the development of merary centrosomes are not sufficient to drive tumor formation. thymic tumors driven by loss of p53. Thus, in either the presence or the absence of p53, centrosome amplification is not a universal centrosome amplification | tumorigenesis | p53 | Plk4 kinase driver of tumor development in mammals. ince their initial description by Theodore Boveri in 1900 (1), Significance Scentrosomes have been recognized as the main microtubule- organizing centers of animal cells and organize bipolar micro- tubule spindle assembly and function during mitosis. To ensure Centrosomes organize the microtubule cytoskeleton in interphase that chromosomes are divided faithfully into the two daughter and mitosis. During mitosis, the centrosomes are important for cells, the number of centrosomes must be precisely controlled. the formation and positioning of the bipolar mitotic spindle on Cells begin the cycle with a single centrosome that duplicates which chromosomes are segregated. The presence of more than exactly once to give rise to two centrosomes that form the poles two centrosomes can drive mitotic chromosome segregation of the mitotic spindle (2, 3). The acquisition of more than two errors and the formation of aneuploid cells. Centrosome amplifi- centrosomes, a state known as centrosome amplification, can cation is a common feature of aneuploid cancer cells, but a long- lead to chromosome segregation errors and subsequent aneu- standing question is whether this is a cause or a consequence of ploidy (4–7). In addition, centrosome abnormalities have been tumor development. To assess this question, we generated mice proposed to lead to alterations in microtubule nucleation and in which centrosome amplification can be induced widely. Despite organization that promote the loss of cell and tissue architecture chronic centrosome amplification, tumorigenesis was not en- observed in cancers. Consistent with this, recent work has shown hanced, demonstrating that an excess of centrosomes is not suf- that supernumerary centrosomes can promote cellular invasion ficient to drive tumor development. in an in vitro model (8). Author contributions: B.V., A.J.H., A.K., O.S., E.F., and D.W.C. designed research; B.V., A.J.H., Centrosome amplification is commonly observed in hemato- A.K., O.S., Y.W., M.M., T.C., and J.B. performed research; B.V., A.J.H., M.H., D.A.S., L.T., S.M.E., logic malignancies and solid tumors, and a clear link exists be- and D.W.C. contributed new reagents/analytic tools; B.V., A.J.H., A.K., O.S., J.B., and D.W.C. tween centrosome amplification and aneuploidy in a wide variety analyzed data; and B.V., A.J.H., A.K., and D.W.C. wrote the paper. of cancer cell lines (6, 7, 9, 10). Furthermore, the presence of The authors declare no conflict of interest. supernumerary centrosomes correlates with increased tumor 1B.V., A.J.H., and A.K. contributed equally to this work. aggressiveness and poor prognosis in human patients (11). Ex- 2Present address: CNRS UMR-5237, Centre de Recherche en Biochimie Macromoleculaire, periments with transplanted larval brain and wing disk tissues in University of Montpellier, Montpellier 34093, France. 3 Drosophila have shown that the presence of extra centrosomes Present address: Department of Molecular Biology and Genetics, Johns Hopkins Univer- sity School of Medicine, Baltimore, MD 21205. can initiate tumorigenesis with (12) or without (13, 14) driving 4Present address: CNRS, UMR-5203, Institut de Génomique Fonctionnelle, INSERM U661, appreciable increases in the level of aneuploidy. University of Montpellier, Montpellier 34094, France. Despite the strong link between centrosome amplification and 5To whom correspondence should be addressed. Email: [email protected]. tumorigenesis, extra centrosomes negatively impact the fitness of This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. mammalian cells and tissues. Induction of centrosome amplifi- 1073/pnas.1519388112/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1519388112 PNAS Early Edition | 1of10 Downloaded by guest on September 28, 2021 A B HEART ATG ATG Transc- CAG promoter H2B-mRFP riptional mouse Plk4 EYFP rabbit β-globin polyA STOP Lox P Lox P 39, F1250 (NT)55, F1 59, F2 36, F2 15, F2 71, F239, F2 10, F2 46 α-RFP + Cre 58 ATG 46 CBB CAG promoter mouse Plk4 EYFP rabbit β-globin polyA Lox P C D E Adenoviral Cre Ad-Cre -++ Non transgenic (NT) Plk4 OE DNA CEP192 Plk4 α-YFP heart muscle spleen lung 125 -+-+ -+-+ α-mRFP 46 α-RFP 46 54 α-Tubulin 55 46 10 μm 10 μm CCB 30 G F NT Plk4 OE DAPI pancreas kidney thymus brain No Ad-Cre Ad-Cre 100 - + -+ -+-+ α-RFP 46 80 54 60 46 CCB Tubulin CEP192 30 40 Ad-Cre Ad-Cre Plk4 OE Cells with > 2 centrosomes (%) 20 0 0102030 Days after Ad-Cre infection 10 μm H Plk4 OE No Ad-Cre Ad-Cre I Plk4 OE J Plk4 OE No Ad-Cre Ad-Cre No Ad-Cre Ad-Cre 20 μm 10 μm 10 μm DAPI Ch 19 DAPI Ch1 Ch19 n= 529 n= 582 30 40 n= 289 n= 19 12 30 n= 219 20 8 n= 23 20 n= 359 n= 215 n= 219 n= 147 n= 364 10 n= 133 4 micronuclei Ch 19 signal 10 % of cells with % nuclei with>2 % of >4N spreads 0 0 0 Adr-Cre - + - + Adr-Cre - + - + Adr-Cre - + - + 39-5 39-3 39-5 39-3 39-5 39-3 Fig. 1. Generation of a mouse model for Cre-inducible Plk4 expression. (A) Schematic of the gene construct used to generate inducible Plk4 OE transgenic mice. A chicken β-actin (CAG) promoter initially directs production of H2B-mRFP. Action of Cre at the two Lox P sites will excise the H2B-mRFP gene and the transcriptional stop cassette, thereby activating Plk4-EYFP expression. (B) Immunoblot of H2B-mRFP in heart tissue lysates from various Plk4 OE mouse founder lines. Numbers indicate the founder line. NT, nontransgenic. The red arrowhead indicates the mRFP signal. (C) Immunoblots to determine the ex- pression of H2B-mRFP in various tissues from the transgenic mouse line 39. (−), control (nontransgenic) mice; (+), Plk4 OE mice. (D) Immunoblot of lysates from MEFs showing the accumulation of the Plk4-EYFP protein. (E) Immunofluorescence images of MEFs at 2 d after transduction with Ad-Cre. Plk4-EYFP (green) is visualized only after Cre expression. Accumulation of supernumerary centrosomes was tracked using CEP192 (red). (Scale bar: 10 μm.) (F) Quantification of centrosome amplification (>2 centrosomes per cell) in MEFs at various times after transduction with Ad-Cre. Points represent the mean of two independent experiments. Error bars indicate SD. (G) Immunofluorescence images showing mitotic figures in Plk4 OE MEFs at 5 d after Ad-Cre transduction or without treatment. (Scale bar: 10 μm.) (H) Representative immunofluorescence image of a chromosome spread of an MEF derived from a Plk4 OE mouse at 5 d after treatment with Ad-Cre.